Replacing SpCas9 residues 1242-1263 with TadA may preserve Cas9 function while adding base-editing capability.
Evidence
This protein-engineering study combined structural, biochemical, and functional analyses of SpCas9 variants, including deletion of residues 1242-1263 and substitution with evolved E. coli TadA.
Caveat
The evidence is a proof-of-concept engineering result, and broader editing precision, delivery, and performance across genomic contexts were not established.
Simplified
The system has emerged as a powerful tool for precise genome editing, with ongoing research focused on enhancing its reliability and expanding its versatility. One effective strategy involves the integration of foreign functional domains into Cas9 to confer new capabilities. However, successful integration requires identification of insertion sites that preserve the protein's structural integrity and function. In this study, we identified a C-terminal region of Streptococcus pyogenes Cas9 (SpCas9), spanning residues 1242-1263, as a viable site for domain replacement. Structural and biochemical analyses of a SpCas9 variant lacking this region confirmed its dispensability for SpCas9 activity. As a proof of concept, we substituted this segment with the evolved E. coli tRNA adenosine deaminase (TadA), a key component of . Functional evaluation of this engineered SpCas9-TadA variant demonstrated deamination efficiency comparable to that of the ABE8e, with the potential to modulate the editing window through linker design. These results highlight the potential of targeted engineering of this region to develop more precise and versatile genome editing tools.
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